PROJECT SUMMARY Difficulty to expand self-renewing human hematopoietic stem cells (HSC) in culture or generate them from human pluripotent stem cells (PSC) has hampered the use of in vitro engineered HSCs for therapeutic purposes. Our data suggest that the failure to induce and maintain the correct transcriptional networks governing HSC self-renewal during in vitro culture compromises the generation/expansion of fully functional human HSC in an in vitro setting. Guided by the transcriptional profile of highly purified human fetal liver (FL) HSC, we sought to identify key transcriptional regulators that govern self-renewal in human HSC, with the long- term goal to develop new strategies to improve the function of in vitro derived hematopoietic cells. We identified MLLT3/AF9, a component of superelongation complex (SEC) as a novel regulator of human HSC stemness. MLLT3 is highly enriched in the self-renewing HSC during human development (FL), pre-natally (cord blood, CB) and in the adult (bone marrow, BM), but becomes downregulated during HSPC differentiation and in vitro culture. Lentiviral knockdown of MLLT3 in human FL and CB HSC resulted in loss of HSC function in vitro and in vivo, while overexpression of MLLT3 greatly improved the ex vivo expansion and engraftment of FL and CB HSPCs, and partially rescued the proliferative potential of hESC-derived HSPCs. An important feature of MLLT3 is that it does not reprogram or transform hematopoietic cells, but only enhances the self- renewal and proliferative potential of properly specific HSCs. We will now examine how MLLT3 co-operates with transcription elongation machinery and epigenetic mechanisms to regulates its target genes and maintain proper HSC stemness program (Aim 1). We will then examine whether rescuing MLLT3 levels in culture by lentiviral overexpression or transient RNA electroporation increases the expansion of in vivo engraftable human HSCs (Aim 2). Finally, we will determine the unique function of a hitherto uncharacterized shorter isoform of MLLT3 that is also highly enriched in human HSCs, and may have an opposing function to the full length MLLT3 (Aim 3). This proposal will help understand how MLLT3 functions as an upstream regulator of ?stemness? in human HSCs, and how its function in regulating HSC fate decisions may be modulated by the different isoforms. These studies will not only increase our knowledge of the fundamental regulatory mechanisms governing human HSC fate decisions, but also pave the way for developing novel approaches for the ex vivo expansion and manipulation of HSC for therapeutic use.